Characterization and properties of protein kinase C from the filamentous fungus Trichoderma reesei.
The Trichoderma reesei pkc1 gene encodes a fungal homologue of the protein kinase C (PKC) family. Using antibodies directed against the nt-sequence-deduced pseudosubstrate domain for identification, Pkc1p was purified by dye-ligand affinity chromatography and Mono Q anion-exchange chromatography. Both the denatured as well as the native enzyme showed an Mr of 116-118kDa, indicating that Pkc1p is a monomer. The enzyme phosphorylates the mutated (A-->S) pseudosubstrate peptide and myelin basic protein, but not histone. Replacing three of the five basic amino acids around the serine acceptor residue resulted in a 25-fold increase in the Km. Pkc1p activity was stimulated by phospholipids, but this stimulation was counteracted by micromolar concentrations of Ca2+. Three proteins (85, 48 and 45 kDa) were identified as preferred endogenous substrates of Pkc1p in vitro. The enzyme was capable of autophosphorylation, and neither phosphorylation nor dephosphorylation in vitro affected the activity of the enzyme. A 116 kDa protein of T. reesei was demonstrated to bind to the N-terminal C2-region of Pkc1p in vitro. These data define Pkc1p as a unique member of the PKC family.
Lendenfeld
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Abteilung für Mikrobielle Biochemie, Institut für Biochemische Technologie und Mikrobiologie, TU Wien, Getreidemarkt 9-172.5, A-1060 Wien, Austria.
Kubicek
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D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D008970:Molecular Weight; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014242:Trichoderma / Q000201:enzymology*
D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D007211:Indoles; D008301:Maleimides; C097993:Pkc1 protein, Trichoderma reesei; D011493:Protein Kinase C; D019311:Staurosporine; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate